Abstract The specificity of glucagon determinations with two pancreatic-type glucagon antisera has been determined by means of gel permeation chromatography before and after treatment of human plasma with acetone and ethanol. Acetone precipitates the interfering high molecular mass substances apparent in the void volume and cross-reacting precursor fragments, while pancreatic glucagon is extracted to slightly more than 50%. Hence, acetone treatment is suggested as a suitable means of obtaining specific glucagon results in single samples. Ethanol also precipitates the high molecular mass substances, but partly extracts the precursor fragments in addition to the glucagon. Ethanol treatment is therefore suggested for chemical diagnosis of the glucagonoma syndrome in which the tumor often produces precursor fragments.